In carrying out biochemical assays and biochemical measurements in clinical tests, there are two methods: one is a method using liquid reagents, and the other is a method using dry reagents (referred to as "dry assay elements"). In both methods, coloration of a reaction system is observed for detection or determination by using light reflectance. Particularly, a rate assay (a method measuring changed amounts at two or more points and the concentration is calculated from the obtained reaction rate) is an assay method generally used for the light absorption measurement using dry assay elements.
In such a method, hemocytes are separated and removed from whole blood and the resulting plasma or serum is used as a sample to be tested. However, the plasma or serum sample is sometimes contaminated by hemoglobin when the sample is prepared from whole blood in which hemolysis of erythrocytes occurred before or during the separation step. As a consequence, the data obtained by this assay become greatly different from the actual data, because the data of the desired component are overlapped with those of hemoglobin (so-called "fogging") and the absorption of hemoglobin changes with time.
Consequently, it is necessary to avoid influence of the changes with time in the absorption of hemoglobin. Some methods have been proposed for this purpose such as a method measuring a blank, a method denaturing hemoglobin physically or chemically to prevent the changes with time (e.g., JP-B-3-58467, the term "JP-B" as used herein means an "examined Japanese patent publication") and a method measuring light absorption at a wavelength of 650 nm or more at which hemoglobin shows less absorption (e.g., JP-A-62-209360, the term "JP-A" as used herein means an "unexamined published Japanese patent application").
Of these methods, the method measuring a blank requires extra labor because the light absorptions of the blank and sample are measured. Particularly, when dry assay elements are used, the blank measurement is difficult because all reagents to be used are combined in one form.
Also, the method denaturing hemoglobin requires complex handling and may denature the desired component to be assayed (for example, enzyme), so that this method is not suitable for the dry assay element whose advantage is easy and simple handling.
The method measuring light absorption at a wavelength of 650 nm or more at which hemoglobin shows less absorption is effective when liquid reagents are used. That is, although absorption of hemoglobin is slightly detectable at a wavelength of 650 nm or more, the fogging of hemoglobin causes no problem when an excessively diluted sample such as the liquid system is measured at a wavelength of small hemoglobin absorption (for example, 680 nm).
However, when a sample is used in an assay without dilution such as the case of dry assay elements, even a small absorption of hemoglobin at a wavelength of 600 nm or more exerts serious problematic influence upon the measured value.